Fusion of viruses with cellular membranes is an essential step for the entry of enveloped viruses, such as HIV-I, HIV-II, RSV, measles virus, influenza virus, parainfluenza virus, Epstein-Barr virus and hepatitis virus, into cells. After having entered the cell the cascade of viral replication may be initiated resulting in viral infection.
HIV is a member of the lentivirus genus, which includes retroviruses that possess complex genomes and exhibit cone-shaped capsid core particles. Other examples of lentiviruses include the simian immunodeficiency virus (SIV), visna virus, and equine infectious anemia virus (EIAV). Like all retroviruses, HIV's genome is encoded by RNA, which is reverse-transcribed to viral DNA by the viral reverse transcriptase (RT) upon entering a new host cell. Influenza viruses and their cell entry mechanisms are described by Bullough, P. A., et al., Nature 371 (1994) 37-43; Carr, C. M., and Kim, P. S., Cell 73 (1993) 823-832; and Wilson, I. A., et al., Nature 289 (1981) 366-373.
All lentiviruses are enveloped by a lipid bilayer that is derived from the membrane of the host cell. Exposed surface glycoproteins (SU, gp120) are anchored to the virus via interactions with the transmembrane protein (TM, gp41). The lipid bilayer also contains several cellular membrane proteins derived from the host cell, including major histocompatibility antigens, actin and ubiquitin (Arthur, L. O., et al., Science 258 (1992) 1935-1938). A matrix shell comprising approximately 2000 copies of the matrix protein (MA, p17) lines the inner surface of the viral membrane, and a conical capsid core particle comprising ca. 2000 copies of the capsid protein (CA, p24) is located in the center of the virus. The capsid particle encapsidates two copies of the unspliced viral genome, which is stabilized as a ribonucleoprotein complex with ca. 2000 copies of the nucleocapsid protein (NC, p7), and also contains three essential virally encoded enzymes: protease (PR), reverse transcriptase (RT) and integrase (IN). Virus particles also package the accessory proteins, Nef, Vif and Vpr. Three additional accessory proteins that function in the host cell, Rev, Tat and Vpu, do not appear to be packaged.
In the case of HIV, viral entry is associated with the HIV envelope surface glycoproteins (Lawless, M. K., et al., Biochemistry 35 (1996) 13697-13708; and Turner, B. G., and Summers, M. F., J. Mol. Biol. 285 (1999) 1-32). In the case of HIV-I, this surface protein is synthesized as a single 160 kD precursor protein, which is cleaved by a cellular protease into two glycoproteins gp-41 and gp-120. gp-41 is a transmembrane protein, and gp-120 is an extracellular protein_which remains non-covalently associated with gp-41 in a trimeric or multimeric form (Hammarskjöld, M.-L., et al., Biochim. Biophys. Acta 989 (1989) 269-280). HIV is targeted to CD4+lymphocytes because the CD4 surface protein acts as the cellular receptor for the HIV-I virus. Viral entry into cells is dependent upon gp-120 binding to the cellular CD4+ receptor molecules while gp-41 anchors the envelope glycoprotein complex in the viral membrane and mediates membrane fusion (McDougal, J. S., et al., Science 231 (1986) 382-385; and Maddon, P. J., et al., Cell 47 (1986) 333-348).
gp41 is the transmembrane subunit that mediates fusion of viral and cellular membranes. The gp41 ectodomain core is a six-helix bundle composed of three helical hairpins, each consisting of an N helix paired with an antiparallel C helix (Chan, D. C., et al., Cell 89 (1997) 263-273; Weissenhorn, W., et al., Nature 387 (1997) 426-430; Tan, K., et al., Proc. Natl. Acad. Sci. USA 94 (1997) 12303-12308). The N helices form an interior, trimeric coiled coil with three conserved, hydrophobic grooves; a C helix packs into each of these grooves. This structure likely corresponds to the core of the fusion-active state of gp41. According to Chan, D. C., et al., Proc. Natl. Acad. Sci. USA 95 (1998) 15613-15617, there is evidence that a prominent cavity in the coiled coil of the HIV type 1 gp41 is an attractive drug target.
It is assumed that the mechanism by which gp-41 mediates membrane fusion may involve the formation of a coiled-coil trimer, which is thought to drive the transition from resting to fusogenic states, as is described, for example, for influenza hemagglutinin (Wilson, I. A., et al., Nature 289 (1981) 366-373; Carr, C. M., and Kim, P. S., Cell 73 (1993) 823-832; Bullough, P. A., et al., Nature 371 (1994) 37-43).
C peptides (peptides corresponding to the C helix) of enveloped viruses, such as DP178 and C34, potently inhibit membrane fusion by both laboratory-adapted strains and primary isolates of HIV-1 (Malashkevich, V. N., et al., Proc. Natl. Acad. Sci. USA 95 (1998) 9134-9139; Wild, C. T., et al., Proc. Natl. Acad. Sci. USA 91 (1994) 9770-9774). A Phase I clinical trial with the C peptide DP178 suggests that it has antiviral activity in vivo, resulting in reduced viral loads (Kilby, J. M., et al., Nature Medicine 4 (1998) 1302-1307). The structural features of the gp41 core suggest that these peptides act through a dominant-negative mechanism, in which C peptides bind to the central coiled coil of gp41 and lead to its inactivation (Chan, D. C., et al., Cell 93 (1998) 681-684).
Within each coiled-coil interface is a deep cavity, formed by a cluster of residues in the N helix coiled coil, that has been proposed to be an attractive target for the development of antiviral compounds. Three residues from the C helix (Trp-628, Trp-631, and Ile-635) insert into this cavity and make extensive hydrophobic contacts. Mutational analysis indicates that two of the N-helix residues (Leu-568 and Trp-571) comprising this cavity are critical for membrane fusion activity (Cao, J., et al., J. Virol. 67 (1993) 2747-2755). Therefore, compounds that bind with high affinity to this cavity and prevent normal N and C helix pairing may be effective HIV-1 inhibitors. The residues in the cavity are highly conserved among diverse HIV-1 isolates. Moreover, a C peptide containing the cavity-binding region is much less susceptible to the evolution of resistant virus than DP178, which lacks this region (Rimsky, L. T., et al., J. Virol. 72 (1998) 986-993). These observations suggest that high-affinity ligands targeting the highly conserved coiled-coil surface, particularly its cavity, will have broad activity against diverse HIV isolates and are less likely to be bypassed by drug-escape mutants.
Fusogenic structures of envelope fusion proteins was shown from influenza, Moloney murine leukemia virus, and simian immunodeficiency virus (cit. in Chan, D. C., Proc. Natl. Acad. Sci. USA 95 (1998) 15613-15617), human respiratory syncytial virus, Ebola, human T cell leukemia virus, simian parainfluenza. It indicates a close relationship between the families of orthomyxoviridae, paramyxoviridae, retroviridae, and others like filoviridae, in which viral entry into target cells is enabled by like transmembrane glycoproteins such as gp41 of HIV-1, hemagglutinin of influenza, GP2 of Ebola and others (Zhao, X., et al., Proc. Natl. Acad. Sci. USA 97 (2000) 14172-14177).
In the state of the art, methods are described for the preparation of peptidic inhibitors (C-peptides) (see, e.g., Root, M. J., et al., Science 291 (2001) 884-888; Root et al. describe peptide C37-H6 which is derived from HIV-1. HXB2 and contains residues 625-661. It was recombinantly expressed as N40-segement with a GGR-linker and a histidine tag, expressed in E. coli and purified from the soluble fraction of bacterial lysates. Zhao, X., et al. describe in Proc. Natl. Acad. Sci. USA 97 (2000) 14172-14177 a synthetic gene of recRSV-1 (human respiratory syncytial virus) which encodes Residues 153-209, a G-rich linker, residues 476-524, Factor Xa cleavage site and a his-tag. Chen, C. H., et al., describe in J. Virol. 67 (1995) 3771-3777 the recombinant expression of the extracellular domain of gp41 synthesized as fusion protein, residues 540-686, fusion to MBP.
A number of peptidic inhibitors, also designated as antifusogenic peptides, of such membrane fusion-associated events are known, including, for example, inhibiting retroviral transmission to uninfected cells. Such peptides are described, for example, by Lambert, D. M., et al., Proc. Natl. Acad. Sci. USA 93 (1996) 2186-2191, in U.S. Pat. Nos. 6,013,263; 6,017,536; and 6,020,459; and in WO 00/69902, WO 99/59615 and WO 96/40191. Further peptides inhibiting fusing associated events are described, for example, in U.S. Pat. Nos. 6,093,794; 6,060,065; 6,020,459; 6,017,536; 6,013,263; 5,464,933; 5,656,480; and in WO 96/19495.
Examples of linear peptides derived from the HIV-I gp-41 ectodomain which inhibit viral fusion are DP-107 and DP-178. DP-107 is a portion of gp-41 near the N-terminal fusion peptide and has been shown to be helical, and it strongly oligomerizes in a manner consistent with coiled-coil formation (Gallaher, W. R., et al., Aids Res. Hum. Retrovirus 5 (1989) 431-440, Weissenhorn, W., et al., Nature 387 (1997) 426-430). DP-178 is derived from the C-terminal region of the gp-41 ecto-domain. (Weissenhorn, W., et al., Nature 387 (1997) 426-430). Although without discernible structure in solution this peptide and constrained analogs therefrom adopt a helical structure, bind to a groove of the N-terminal coiled-coil trimer of gp41 and thus prevent the gp41 to transform into the fusogenic state (Judice, J. K., et al., Proc. Natl. Acad. Sci. USA 94 (1997) 13426-13430).
Such short-chain peptides usually are prepared by chemical synthesis. Chemical synthesis is described, for example, by Mergler, M., et al., Tetrahedron Letters 29 (1988) 4005-4008 and 4009-4012; Andersson, L., et al., Biopolymers 55 (2000) 227-250; and by Jones, J. H., J. Pept. Sci. 6 (2000) 201-207. Further methods are described in WO 99/48513.
However, chemical peptide synthesis suffers from several drawbacks. Most important is racemization, which results in insufficient optical purity. In peptide chemistry, racemization also means epimerization at one of several chirality centers. If only 1% racemization occurs for a single coupling step, then at 100 coupling steps only 61% of the target peptide would be received (Jakubke, H. D., Peptide, Spektrum Akad. Verlag, Heidelberg (1996), p. 198). It is obvious that the number of impurities increases with growing chain length and their removal is more and more difficult and costly.
Chemical synthesis on large scale is limited by high costs and lack of availability of protected amino acid derivatives as starting materials. On the one hand, these starting materials should be used in excess to enable complete reactions, on the other hand, their use should be balanced for cost reasons, safety and environmental aspects (Andersson et al., Biopolymers 55 (2000) 227-250).
Peptides may also be produced by recombinant DNA technology. Whereas recombinant production of soluble proteins of chain lengths of more than 50 amino acids is known from the state of the art, the production of peptides with fewer than 50 amino acids suffers from several drawbacks (Doebeli, H., et al, Protein Expression and Purification 12 (1998) 404-414). Such short or medium chain peptides are usually not stably expressed. They are attacked by intrinsic peptidases and degraded. This may result from their small size or lack of highly ordered tertiary structure (WO 00/31279). Other authors have found that recombinant production of peptides requires a minimum chain length of 60 to 80 amino acids for a stable expression, and it is further common knowledge that such peptides are produced as soluble peptides and not as inclusion bodies (see, e.g., van Heeke, G., et al., Methods in Molecular Biology 36 (1994) 245-260, eds. B. M. Dunn and M. W. Pennington, Humana Press Inc., Totowa, N. J.); and Goldberg et al., Maximizing Gene Expression (1986), pp. 287-314, eds. Reznikoff, W., and Gold, L., Butterworks, Stoneham, Mass.). Recombinant production of shorter peptides is especially not successful because if such peptides are expressed in prokaryotes, they remain soluble and are immediately degraded by prokaryotic peptidases. To avoid this problem, according to common knowledge, such peptides are expressed as large (more than 150 to 200 amino acids) fusion proteins, whereby the fusion tail either renders the fusion protein fairly soluble and avoids the formation of inclusion bodies or the fusion tail is a protein which forms during recombinant expression in prokaryotes, inclusion bodies, and therefore fusion protein consisting of such fusion tail and the desired short peptide will also form inclusion bodies during overexpression in prokaryotes. A great disadvantage of such methods is that the molecular weight of the fusion tail is considerably higher than the molecular weight of the desired peptide. Therefore, the yield of the desired peptide is very low and the excess of cleaved fusion tail has to be separated of.
Lepage, P., et al., in Analytical Biochemistry 213 (1993) 40-48, describe recombinant methods for the production of HIV-1Rev peptides. The peptides are expressed as fusion proteins with the synthetic immunoglobulin type G (IgG) binding domains of Staphylococcus aureus protein A. The peptides have a length of about 20 amino acids, whereas the IgG-binding part has a length of about 170 amino acids, so that the expressed fusion protein has an overall length of about 190 amino acids. This fusion protein is expressed, secreted in soluble form in the medium, and purified by affinity chromatography. The authors reported that with this method it might be possible to produce recombinant protein in an amount of hundreds of milligrams per liter of culture. However, this methodology is limited due to alternative processing within the signal peptide sequence and several post-translational modifications of the fused proteins as well as of the cleaved peptides. Assuming an average molecular weight of an amino acid of 110 Daltons, the desired peptides have a molecular weight of about 2,000 to 5,000 Daltons, whereas the fusion tail has a length of at least 170 amino acids (about 19,000 D), if the IgG binding domains of Staphylococcus aureus protein A is used as such a fusion tail. Therefore, only 10 to 25% of the recombinantly produced protein is the desired peptide.
EP 0 673 948 describes the recombinant production of a gp41 peptide as a fusion protein with β-galactosidase using the expression vector pSEM3 (Knapp, S., et al., BioTechniques 8 (1990) 280-281). This fusion protein contains a large part of β-galactosidase gene, encoding the N-terminal 375 amino acids and additional 23 codons of a polylinker sequence.
Further examples and methods for the recombinant production of small peptides via large fusion proteins in E. coli are described by Uhlen and Moks, “Gene Fusions For Purposes of Expression, An Introduction” in Methods in Enzymology 185 (1990) 129-143, Academic Press. In regard to the production via the “inclusion body” way, Uhlen and Moks refer to large fusion products involving fusion parts like trpE, cII and again β-galactosidase. Ningyi, L., et al., Gaojishu Tongxun 10 (2000) 28-31 describe the recombinant expression of p 24 gag gene in E. coli. 